Highly sensitive, reliable and practical detection of Cyclooxygenase-2 (COX-2) as an important enzyme in pain biomarkers, inflammation, and cancer cell proliferation is of great importance. This study demonstrates the feasibility of developing nanobiosensor (or biosensor) platforms for sensitive COX-2 biomarker detection from phosphate buffer saline (PBS) and human serum samples. Highly porous polyaniline nanofibers were fabricated in four different average diameters using electrospinning process. They were then integrated as the main sensing component into interdigitated gold microelectrodes to develop label-free electrochemical nanobiosensors. The performance of the nanobiosensors was evaluated in comparison with a plain control biosensor using electrochemical impedance spectroscopy (EIS). Significant improvement was observed in the sensitivity of the nanofiber-based biosensors revealing the remarkable capability of electrospun polyaniline nanofiber in robust and rapid detection of the COX-2 biomarker. The fabricated nanofiber-based biosensors were able to detect the target antigen with concentrations as low as 0.01 pg/ml in PBS and human serum samples, respectively. High sensitivity, as well as remarkable selectivity towards COX-2 analyte, suggested the excellent potential of this nanofiber-based platform for reliable COX-2 analyte detection.